CN112301115B - FGFRs gene mutation detection method based on high-throughput sequencing and probe sequence - Google Patents
FGFRs gene mutation detection method based on high-throughput sequencing and probe sequence Download PDFInfo
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Abstract
The invention relates to a detection method and a probe sequence of FGFRs gene mutation based on high-throughput sequencing, which comprises the following steps: 1) Co-extracting sample genome DNA and total RNA; 2) Fragmenting the genome DNA and recovering the fragmented DNA; 3) Performing total RNA interruption and/or primer hybridization according to the total RNA quality control condition; 3) Synthesizing a first strand of cDNA; 4) Synthesizing a second cDNA chain; 5) Mixing the fragmented DNA with cDNA to construct a mixed library; 6) Hybridizing and capturing by using a capture probe; 7) Capturing, amplifying and purifying the library; 8) High throughput sequencing and mutation analysis. The invention can detect single base mutation, insertion deletion mutation, fusion mutation and relative expression analysis of FGFR1, FGFR2, FGFR3 and FGFR4 genes.
Description
Technical Field
The invention relates to a FGFRs (FGFR 1, FGFR2, FGFR3 and FGFR 4) gene mutation detection method developed based on a high-throughput sequencing platform.
Background
Fibroblast Growth Factor Receptors (FGFRs) are highly conserved, widely distributed transmembrane tyrosine kinase receptors. They are involved in development, differentiation, cell survival, migration, angiogenesis and carcinogenesis. At present, a plurality of FGFR targeted drugs are being developed by top drug enterprises at home and abroad, including first-generation drugs such as Erdafitinib (erda tinib), BGJ398, AZD4547, BLU-554 and the like, wherein the Erdafitinib is already sold on the market, meanwhile, the second-generation FGFR targeted drug TAS-120 after first-generation drug resistance is also in the clinical evaluation stage, and the requirement of the concomitant diagnosis clinical detection on the drug target of the targeted drugs is vigorous day by day. In humans, there are four tyrosine kinase receptors typical for FGFR (FGFR 1, FGFR2, FGFR3, FGFR 14), and mutation profiles of FGFR in 4853 patient samples from various cancers, including mutation, fusion, and amplification, were described using next generation sequencing technologies in clinical cancer research journal in 2015. Of the 4853 cancers sequenced, 360 FGFR mutations were observed in 343 cases from this study, involving 17 cancer species in total, with a total incidence of 7.1%. Among 360 FGFR mutations, point mutation and insertion deletion mutant point genes account for about 26%, and gene fusion mutation accounts for about 8%.
Based on the diversity of the mutation types of the FGFRs genes, including point mutation, insertion deletion mutation, fusion mutation, amplification/overexpression mutation, the clinical detection requirements are also diverse, and a method capable of detecting multiple mutation types of FGFRs is needed; while being limited by sample size, low sample quality, and economics of the end-point detection application. The existing FGFRs gene mutation detection method has a plurality of defects.
The FGFR2 gene fusion mutation detection technology based on Fluorescence In Situ Hybridization (FISH) is characterized in that a 5 'end probe set and a 3' end probe set are respectively designed according to the upstream and the downstream of a FGFR2 gene, different fluorescent groups are marked, and whether fusion mutation occurs or not is judged by observing the separation degree of fluorescence signals of in situ hybridization under a microscope. The method can be only used for identifying whether the FGFR2 gene has fusion mutation, can not determine the fusion partner gene and the specific breakpoint position, and can not detect other mutation types such as point mutation, insertion deletion mutation and the like of the FGFR gene.
Based on independent amplicon relying on DNA sample or targeted capture high-throughput detection technology, by designing an amplification primer or a capture probe of a CDS region of FGFRs gene, the detection of FGFRs gene point mutation and insertion deletion mutation can be realized, but for fusion mutation detection and amplicon targeted sequencing technology, the amplification primer can be designed only according to known fusion break points, the detection method cannot be applied to DNA samples with unknown break points in fusion occurrence in intron regions, and certain requirements are also made on sample quality; for the targeted capture sequencing technology, theoretically, the capture probe needs to cover all the intron regions of the genes to cover all the fusion mutation types of the FGFRs genes, and compared with the detection of the CDS region, the detection region is enlarged by 15 times, the expected sequencing data volume and the sequencing cost are also increased by 15 times, so that the detection cost is too high, and the economy is poor.
Based on a targeted capture high-throughput detection technology which respectively depends on a DNA sample and an RNA sample, an amplification primer or a capture probe can be designed for a DNA exon region to realize the detection of point mutation and insertion deletion mutation, and for an amplicon targeted sequencing technology which adopts the RNA sample to carry out fusion mutation detection, the amplification primer can be designed only according to known fusion breakpoints to realize the detection of known fusion mutation types, but unknown fusion mutation cannot be detected; for the targeted capture sequencing technology, the detection of the known fusion mutation and the unknown fusion mutation can be realized, but 2 sample types need to be respectively subjected to hybridization capture steps, so that the detection operation is complex, the detection cost is doubled, and the applicability and the economy are poor.
In summary, the current FGFRs mutation detection methods are not mature enough, and the available detection methods are limited by the technical principle of being not applicable, incomplete in mutation type detection, too high in detection cost or too complex in detection operation, and cannot meet the increasing detection requirements, so that a method which is comprehensive in mutation type detection, low in detection cost, simple in detection steps and capable of meeting the clinical detection requirements of FGFRs is urgently needed.
Disclosure of Invention
The invention mainly aims to provide a FGFR1, FGFR2, FGFR3 and FGFR4 gene mutation detection method developed based on a high-throughput sequencing platform. The invention is realized by the following technical scheme:
according to the sequence of the coding regions of FGFR1, FGFR2, FGFR3 and FGFR4, a capture probe and a hybridization capture elution system for detecting the point mutation, the insertion deletion mutation, the fusion mutation and the relative expression analysis of the genes of FGFR1, FGFR2, FGFR3 and FGFR4 are designed and screened out through a large number of long-term experimental researches, and the capture probe and the hybridization capture elution system comprise but are not limited to SEQ ID No:1 to 20.
Probe sequence of SEQ ID No.
Note: MT/C/G-Biotin is Biotin modified base
According to the coding region sequence and the intron sequence of each housekeeping gene, through long-term massive database research, experimental research, design and screening of capture probes comprising TBC1D10B, VCP, VPS29, RAB7A, TBP, EMC7, CHMP2A, CHMP2A, CHMP2A, POLR A, ABCF, PSMB2, MRPL13, UBB, OAZ1, GPI, PSMB4, HMBS, GAPDH, STK11IP, G6PD, SNRPD3, HPRT1 and other housekeeping gene exon boundary region capture probes, as well as these housekeeping gene intron region capture probes and hybridization elution systems, including but not limited to SEQ ID No: 21-30.
And establishing a library construction system for constructing a DNA and cDNA mixed library, and realizing the end repair and joint connection reaction of the broken DNA and cDNA in the same reaction system.
A FGFR1, FGFR2, FGFR3 and FGFR4 gene mutation detection method developed based on a high-throughput sequencing platform is designed, and comprises the following steps:
1) Synthesizing the probe, and labeling the 5 'or 3' end of the probe sequence with biotin;
2) Co-extracting genome DNA and total RNA of tissue FFPE samples and controlling quality:
3) Genomic DNA fragmentation: adopting confocal ultrasound or DNA digestive enzyme to segment the genome DNA
4) Performing total RNA interruption and/or primer hybridization according to the quality control condition of the total RNA;
5) cDNA Synthesis: synthesizing a first cDNA chain by using the total RNA as a template, and synthesizing a second cDNA chain by using the first cDNA chain as a template;
6) Constructing a DNA and cDNA mixed library: taking the fragmented DNA product, mixing the fragmented DNA product with the purified cDNA product, and adding a terminal repair buffer solution and a terminal repair enzyme mixture to carry out terminal repair reaction; after the reaction is finished, adding a joint, a connection buffer solution, an enhancer and ribozyme-free water for connection reaction; after purifying the product, adding a tag primer to perform library amplification reaction, purifying to obtain a DNA and cDNA mixed library, and performing quantification and quality control on the library;
7) Hybridizing and capturing; taking the constructed DNA and cDNA mixed library, adding hybridization buffer solution, FGFR1, FGFR2, FGFR3 and FGFR4 genes and a quality control reference gene capture probe for hybridization and capture;
8) Amplifying and purifying the captured library; adding an amplification primer and an amplification enzyme into the captured recovery product to perform library amplification, and purifying the recovery product;
9) High-throughput sequencing: and performing high-throughput sequencing after quantification and quality control of the amplified library.
10 Analysis of sequencing results: and (3) carrying out sequence analysis on the sequencing result, and outputting single base mutation, insertion deletion mutation, fusion mutation information and relative expression amount information of the FGFR1, FGFR2, FGFR3 and FGFR4 genes.
According to the invention, genomic DNA and RNA are adopted to construct a library in the same system and perform hybrid capture, so that all point mutations, insertion deletion mutations and all known or unknown fusion types of FGFR1, FGFR2, FGFR3 and FGFR4 gene coding regions can be detected, and meanwhile, the relative expression quantity information of FGFR1, FGFR2, FGFR3 and FGFR4 genes can be obtained, and the FGFRs gene detection requirements can be completely met. The DNA and RNA samples are adopted for parallel tube detection, the maximum advantages of the technology are exerted, the detection accuracy and sensitivity are ensured, the detection steps are simpler and more convenient, and the detection cost is lower.
Compared with the existing detection products, the invention has the main beneficial effects that:
1. the detection of mutation types is comprehensive: the detection can output all point mutation, insertion deletion mutation and fusion mutation of the FGFRs gene coding region, and can output the relative expression quantity information of the FGFRs gene.
2. DNA/RNA co-detection: the DNA/RNA parallel tube detection is realized, the accuracy and the sensitivity of the detection mutation detection are determined, and meanwhile, the operation steps are simplified and the detection cost is reduced.
3. Detecting unknown fusion: all known and unknown fusion mutation types of FGFRs can be detected.
Drawings
The invention is further illustrated by the following figures and examples.
FIG. 1 is a flow chart of the detection of the present invention
In FIG. 2: internal reference gene design picture-according to mRNA sequence, designing capture probe of exon boundary region for quality control and quantitative cDNA library derived reads; the following: based on the genome sequence, capture probes for intron regions were designed for quality control of DNA library-derived reads.
Detailed Description
Example 1
The standard substance is the core for evaluating the detection capability of different detection methods, and different mutation detection systems can be evaluated fairly and fairly only by constructing the standard substance with accurate mutation sites, types and mutation frequencies. The invention takes FGFRs gene mutation as a research object, and evaluates the capability of the invention for detecting FGFRs gene mutation by constructing FGFRs gene point mutation and fusion mutation, wherein the specific mutation types are shown in Table 2.
(1) Preparation of standards
Point mutation standard substance, using tumor cell line (HCT-116, ATCC CCL-247) with known FGFRs mutation information, and adopting corresponding wild type cell line (HEK-293T, ATCC CRL-11268) to dilute into mixed cell line with mutation frequency of 7.5%,5%,2.5%,1% point mutation standard substance; the fusion standard substance is prepared by using commercial standard substance RNA (CBP 20080R and CBP 20105R) of known FGFR2 fusion mutation information, sequentially diluting the commercial standard substance RNA with corresponding wild type standard substance RNA to fusion mutation copy numbers of 1000 copies/100 ng, 750 copies/100 ng, 500 copies/100 ng, 250 copies/100 ng and 100 copies/100 ng, and mixing the DNA of the fusion standard substance with wild type DNA in the same mixing ratio. The FGFRs gene mutation detection method is based on the invention, and the prepared point mutation and fusion mutation sensitivity standard substance is subjected to library construction, sequencing and analysis of the detection conditions of point mutation and fusion.
(2) Sensitivity standard detection
1. Extracting genome DNA and RNA of the prepared point mutation sensitivity standard sample and controlling quality: using DNA and RNA co-extraction kit (Thermo Fisher, magMAX) TM FFPE DNA/RNA Ultra Kit, cat # A31881 or Covaris, truXTRACFFPE total NA Kit, cat No. 520220) requires that the total amount of DNA is not less than 100ng, and the total amount of RNA is not less than 100ng (DV 200 is greater than or equal to 30%) or 500ng (DV 200 is less than 30%).
2. DNA fragmentation: 100ng of DNA qualified in quality control is taken, and Covaris M220 or a similar instrument or KAPA fragment Kit for enzymic Fragmentation (cat No.: KK 8602) is adopted for genome DNA Fragmentation, so that the length of the fragmented DNA is mainly distributed between 150 and 350 bp. And purifying and recovering the fragmented DNA by using purified magnetic Beads (such as AMPure XP Beads), and finally eluting by using 22ul of purified water for later use.
3. RNA disruption and primer hybridization:
1) For RNA samples with DV200 content of 30% or more, 100ng of RNA samples qualified for detection are taken, and 4. Mu.l of 5 Xfirst strand synthesis buffer (components of the kit for establishing a library: NEBNext First Strand Synthesis Reaction Buffer (5X)) and 1. Mu.l of 6bp random primers (synthesized) and made up to a total volume of 18ul with nucleic-free H2O, placed on a PCR instrument, and subjected to cleavage and primer hybridization reactions according to the following procedure: 94 ℃ for 8min (when DV200 is greater than or equal to 70%, increase to 15 min), 4 ℃ for maintenance, and hot cover 105 ℃;
2) For RNA samples with DV200 less than 30% of RNA samples, 500ng of RNA samples were taken, 1. Mu.l of 6bp random primer (synthesized) was added, and the total volume was made up to 14ul with nucleic-free H2O, and placed on a PCR instrument for primer hybridization according to the following procedure: 65 ℃ for 5min,4 ℃ hold, hot lid 105 ℃.
4. First strand cDNA Synthesis:
for RNA samples with DV200 at 30% or more, 2. Mu.l of First Strand synthetase Mix (library building kit components: NEBNext First Strand Synthesis Enzyme Mix) was added to the disrupted RNA in a total volume of 20. Mu.l. The reaction was carried out on a PCR instrument according to the following procedure: 10 cycles of 25 ℃ for 10min,42 ℃ for 30min, (60 ℃ for 2min,42 ℃ for 2 min), 70 ℃ for 15min,4 ℃ for 4 ℃, and the temperature of a hot cover is not lower than 80 ℃.
2) For RNA samples with DV200 less than 30%: to the RNA to which the primer hybridization had been carried out, 4ul of 5 Xfirst Strand Synthesis Buffer (pooling kit component: NEBNext First Strand and Synthesis Reaction Buffer (5X)) and 2. Mu.l of First Strand Synthesis Enzyme mixture (pooling kit component: NEBNext First Strand and Synthesis Enzyme Mix (Mix)) were added in a total volume of 20. Mu.l, and placed on a PCR apparatus, and the Reaction was carried out according to the following procedure: 10 cycles of 25 ℃ for 10min,42 ℃ for 30min, (60 ℃ for 2min,42 ℃ for 2 min), 70 ℃ for 15min,4 ℃ for 4 ℃, and the temperature of a hot cover is not lower than 80 ℃.
5. Second strand cDNA Synthesis: to the product of the first Strand Synthesis, 8. Mu.l of 10 Xsecond Strand Synthesis Buffer (pooling kit components: NEBNext Second Strand and Synthesis Reaction Buffer with dUTP (10X)), 4. Mu.l of Second Strand Synthesis Enzyme mixture (pooling kit components: NEBNext Second Strand and Synthesis Enzyme Mix) and 48. Mu.l of ribozyme-free water were added in a total volume of 80. Mu.l, and placed on a PCR apparatus, and the Reaction was carried out according to the following procedure: 60min at 16 ℃, keeping at 4 ℃ and keeping the temperature of the hot cover not higher than 40 ℃, or not covering the hot cover; after completion of the reaction, 176. Mu.l of purified magnetic beads (Beckman Coulter, AMPure XP magnetic beads, cat. No.: A63881) were added and purified, and 33. Mu.l of 0.1 XTE buffer was used for elution, and 31. Mu.l of the product was recovered.
6. Construction of a DNA and cDNA library mixture, 20ul of fragmented DNA and 30ul of purified cDNA product were mixed, 7ul of terminal repair Buffer (library construction kit component: NEBNext Ultra II End Prep Reaction Buffer) and 3 ul of terminal repair Enzyme mixture (library construction kit component: NEBNext Ultra II End Prep Enzyme Mix) were added, and the Reaction was carried out in a total volume of 60 ul according to the following procedure: 30min at 20 ℃, 30min at 65 ℃ and keeping at 4 ℃; after the reaction, 1. Mu.l of linker, 30. Mu.l of Ligation buffer (component of a library building kit: NEBNext Ultra II Ligation Master Mix) and 1. Mu.l of Enhancer (component of a library building kit: NEBNext Ligation Enhancer) were added to the final repair product, and the reaction was carried out according to the following procedure: keeping at 20 deg.C 30min and 4 deg.C without covering a hot cover; after completion of the reaction, 86. Mu.l of purified magnetic beads (Beckman Coulter, AMPure XP magnetic beads, cat. No.: A63881) were added and purified, and then eluted with 19. Mu.l of 0.1 XTE buffer, and 17. Mu.l of the product was recovered.
7. Library amplification: 17ul of the product obtained above was taken and added with 25 ul of amplification reaction solution (KAPA, 2 XKAPA Hifi Ready mix, cat # KK 2806) and 8ul of 25uM of tagged primers (primers were synthesized with reference to Illumina standard sequence, each pair of primers was labeled with a different index, and different samples were labeled with different primers) in a total volume of 50 ul. Library amplification was performed according to the following procedure:
after the reaction, 40. Mu.l of purified magnetic beads (Beckman Coulter, AMPure XP magnetic beads, cat. No.: A63881) were added and purified, and then eluted with 32. Mu.l of nuclease-free water, whereby 30. Mu.l of the product was recovered. The purified DNA and cDNA mixed library product is quantified, and the library yield is required to be more than or equal to 450ng.
8. Hybridization and capture: taking the constructed DNA and cDNA mixed library, adding hybridization buffer solution, FGFR1, FGFR2, FGFR3 and FGFR4 genes and a quality control reference gene capture probe (shown as SEQ ID NO: 1-30) for hybridization and capture, and specifically comprising the following steps:
1) Concentrating the library, taking 400-1000 ng of the purified DNA and cDNA mixed library, adding 7 mul of sealant, placing in a vacuum concentrator, setting the temperature at 60 ℃, centrifuging until the liquid in the tube is evaporated to dryness (about 30-60 minutes), and eluting by using 10 mul of hybridization buffer solution after the liquid is evaporated to dryness. The fractions were also concentrated using Ampure XP Beads, mixed with 2 bead volumes, washed with 80% ethanol and eluted with 10. Mu.L of hybridization buffer.
2) And (3) hybridizing, adding 5 mu L of capture probe into the eluted hybridization solution, placing the sample tube on a PCR instrument, and performing hybridization by setting the following program: 10 minutes at 95 ℃; at 47 deg.c for 16-20 hr.
3) Capture, 10. Mu.L Dynabeads MyOne TM Washing Streptavidin T1 magnetic beads for 3 times by using a magnetic bead washing buffer solution, transferring all hybridized reaction solution into a washed magnetic bead reaction tube, blowing, uniformly mixing, placing into a PCR instrument,incubate at 48 ℃ for 45 minutes, and take out and mix well every 15 minutes.
4) Washing, after the capture is finished, resuspending the magnetic beads in the capture reaction solution by using 1 × washing buffer (2) preheated at 48 ℃, transferring the magnetic beads into a 1.5ml centrifuge tube and washing; then adding preheated 1 Xwashing buffer (1), 200. Mu.L, placing on a constant temperature metal oscillator, incubating at 48 ℃ for 5 minutes and washing for 2 times; then, a pre-heated 1 XWash buffer (2. Mu.L) was added thereto, and the mixture was washed 1 time at 48 ℃ for 5min, followed by 200. Mu.L of 1 XWash buffer (3) and 200. Mu.L of 1 XWash buffer (4), respectively. After washing was completed, the captured product was recovered with 20. Mu.L of purified water.
9. Post-capture amplification: mu.L of the product after hybridization capture was taken, and then 29. Mu.L of the amplification reaction solution (2) and 1. Mu.L of polymerase were added thereto in a total volume of 50. Mu.L. Library amplification was performed according to the following procedure:
after the reaction, 50. Mu.l of purified magnetic beads (Beckman Coulter, AMPure XP magnetic beads, cat. No.: A63881) were added and purified, and then eluted with 32. Mu.l of 0.1 XTE buffer, and 30. Mu.l of the product was recovered.
10. Quality control of the library: the concentration is more than 1 ng/. Mu.l, and the fragment is qualified between 200 and 500 bp.
11. Sequencing on a computer: library sequencing was performed using the Illumina high throughput sequencing platform with a sequencing read length of 2 × 150bp.
12. And (3) data quality control: the sequencing data Q30 of the sample is required to be greater than or equal to 75%; coverage (Coverage) should be greater than or equal to 98%; the average effective depth (SSBCDepth, i.e. the corrected depth of the single strand) should be not less than 500 ×; the area coverage depth of the CDS area is more than 100 multiplied by the area ratio (SSBC 100) is more than or equal to 99 percent; the RNA-derived reads should cover a depth of 250 × (RNADP); the depth of coverage (DNADP) of the DNA-derived reads should be 500X or more.
13. And (3) data analysis: the mansion Ai Deren FGFR1, FGFR2, FGFR3 and FGFR4 gene mutation detection data analysis system (ADXGFFR-tMut) analyzes the detection data and outputs the information of single base mutation, insertion deletion mutation and fusion mutation of the FGFR1, FGFR2, FGFR3 and FGFR4 genes and the information of relative expression amount of the FGFRs genes.
14. And (4) conclusion: this example allowed the stable detection of SNV at a minimum mutation frequency of 5% and fusion mutations at low to 250 copies in 100ng RNA. The limit of detection by various sensitivity standards was 5% for point mutations and 250 copies/100 ng for fusion mutations.
Example 2
In order to demonstrate the accuracy of the detection of the FGFRs fusion mutation of the present invention, genomic DNA and total RNA of 122 intrahepatic bile duct cancer FFPE samples (12 FGFR2 gene fusion positive samples and 110 FGFR2 gene fusion negative samples detected by FISH) with known detection results of the FGFR2 gene fusion FISH platform were extracted, and the detection was performed by using the method for detecting FGFR1, FGFR2, FGFR3, and FGFR4 gene mutations of the present invention. And analyzing and comparing the data obtained by sequencing. The FGFR2 fusion mutation detection accuracy and the capability of detecting other FGFRs gene mutations are embodied, and the detection statistical results are shown in the following table 2.
And (4) conclusion: for FGFR2 fusion positive and negative detection, the detection result of the invention is completely consistent with the FISH detection result; in addition, the invention can also accurately detect the position of a chaperone gene and a breakpoint fused with FGFR2, and can also detect the fusion condition of other FGFRs genes.
Table 2:122 cases of intrahepatic bile duct cancer FFPE sample FGFRs fusion detection results
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aaacattctt ttacatgttt ggtacttgtt cagctttatt caagtggaat ttctgggtc 59
<210> 24
<211> 59
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 24
aaagaatgtc ttgattgtgg aagatataat tgacactggc aaaacaatgc agactttgc 59
<210> 25
<211> 59
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 25
tatatataaa cttatatatt tatgtagtta aaattaaaag taattaaaaa tttagttaa 59
<210> 26
<211> 59
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 26
tctggaatat actcatctgg aaaaagatgc aacctttcca tcattgttct tctgtgaag 59
<210> 27
<211> 59
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 27
tctcagatga ggaaactgag gccctgtgaa agtacatgag ctgctatcag tctcaggat 59
<210> 28
<211> 59
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 28
gcgcaggagt gcaaaaaaga tctccccgtc cagctgaatg gcctccagcc ctgcactgt 59
<210> 29
<211> 59
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 29
aaggcaagct tttcatgcag catttagcct ttttgtccca gagcatctga aaactgaat 59
<210> 30
<211> 59
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 30
cgccgaatat aatcccaagc ggtttgctgc ggtaatcatg aggataagag agccacgaa 59
Claims (8)
1. A FGFRs gene mutation detection method based on high-throughput sequencing, wherein the FGFRs gene comprises FGFR1, FGFR2, FGFR3 and FGFR4, and is characterized by comprising the following steps:
1) Co-extracting genome DNA and total RNA of tissue FFPE samples and controlling quality:
2) Fragmenting the genomic DNA;
3) Performing total RNA interruption and/or primer hybridization according to the quality control condition of the total RNA;
4) And (3) cDNA synthesis: synthesizing a first cDNA chain by using the total RNA as a template, and synthesizing a second cDNA chain by using the first cDNA chain as a template;
5) Constructing a DNA and cDNA mixed library: taking the fragmented DNA product, mixing the fragmented DNA product with the purified cDNA product, and adding a terminal repair buffer solution and a terminal repair enzyme mixture to carry out terminal repair reaction; after the reaction is finished, adding a joint for connection reaction; after purifying the product, adding a label primer to perform library amplification reaction, purifying to obtain a DNA and cDNA mixed library, and performing quantification and quality control on the library;
6) Hybridizing and capturing; taking the constructed DNA and cDNA mixed library, and adding hybridization buffer solution, FGFR1, FGFR2, FGFR3 and FGFR4 genes and a quality control reference gene capture probe for hybridization and capture; the FGFR1, FGFR2, FGFR3 and FGFR4 gene capture probes comprise capture probes of complete CDS regions of FGFR1, FGFR2, FGFR3 and FGFR4 genes, and are used for capturing FGFR gene sequences so as to detect single base mutation, insertion deletion mutation, fusion mutation and overexpression analysis of the FGFR genes;
the FGFR1, FGFR2, FGFR3 and FGFR4 gene capture probes comprise SEQ ID NO 1-20;
the reference gene capture probe comprises TBC1D10B, VCP, VPS29, RAB7A, TBP, EMC7, CHMP2A, POLR2A, ABCF, PSMB2, MRPL13, UBB, OAZ1, GPI, PSMB4, HMBS, GAPDH, STK11IP, G6PD, SNRPD3 and HPRT1, wherein the capture probe of the exon border region of the housekeeping gene is used for controlling the sequence derived from cDNA and homogenizing the gene table among samples, and the capture probe of the intron region of the housekeeping gene is used for controlling the sequence derived from DNA;
7) Amplifying and purifying the captured library; adding an amplification primer and an amplification enzyme into the captured recovery product for library amplification, and purifying the recovery product;
8) High-throughput sequencing: performing high-throughput sequencing after quantifying and quality controlling the amplified library;
9) And (3) analyzing a sequencing result: and (3) carrying out sequence analysis on the sequencing result to obtain single base mutation, insertion deletion mutation and fusion mutation information of FGFR1, FGFR2, FGFR3 and FGFR4 genes and relative expression amount information of FGFRs genes.
2. The method for detecting mutations in FGFRs genes according to claim 1, wherein the method comprises the following steps: the co-extraction in the step 1) is the co-extraction of genome DNA and total RNA, and the extracted sample is subjected to single nucleic acid extraction to respectively obtain the genome DNA and the total RNA.
3. The method for detecting mutations in FGFRs genes according to claim 1, wherein the method comprises the following steps: the sample in the step 1) is a sample with serious RNA degradation, and comprises a formalin-fixed paraffin-embedded sample.
4. The method for detecting mutations in FGFRs genes according to claim 1, which is based on high throughput sequencing, wherein: in step 3), for RNA samples with DV200 being more than or equal to 30%, 100ng of RNA samples qualified for detection is taken, 4 muL of 5 Xthe first strand synthesis buffer and 1 muL of random primer are added, and nucleic-free H is used 2 And (3) filling O to a total volume of 18 mu L, placing on a PCR instrument, and performing interruption and primer hybridization reaction according to the following program: 94 ℃ for 8min,4 ℃ for maintenance and 105 ℃ for hot cover;
for RNA samples with DV200 less than 30% of RNA samples, 500ng of RNA sample is taken, 1. Mu.L of random primer is added, and nucleic-free H is used 2 And (3) filling O to a total volume of 14 mu L, placing on a PCR instrument, and carrying out primer hybridization reaction according to the following procedures: 65 ℃ for 5min,4 ℃ hold, hot lid 105 ℃.
5. The method for detecting mutations in FGFRs genes according to claim 1, wherein the method comprises the following steps: in the step 5), the construction of the DNA and cDNA mixed library is carried out in the same reaction system, and the end repair reaction, the joint connection reaction and the library amplification reaction of the label primer pair of the DNA fragment and the cDNA fragment are carried out simultaneously.
6. The method for detecting mutations in FGFRs genes according to claim 1, wherein the method comprises the following steps: in the step 6), the target region DNA fragments in the constructed DNA and cDNA mixed library are hybridized by adopting a biotin-labeled capture probe in the same reaction system, and the target region DNA fragments are captured and separated by adopting streptavidin-labeled magnetic beads, so that the target region DNA fragments in the DNA and cDNA mixed library are separated and enriched in the same reaction system.
7. The method for detecting mutations in FGFRs genes according to claim 1, wherein the method comprises the following steps: the capture probes for the intron regions of the housekeeping genes include SEQ ID NOS: 21-30.
8. A probe sequence combination for detecting FGFRs gene mutations, comprising SEQ ID NO: 1-SEQ ID NO:30.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109637587A (en) * | 2019-01-18 | 2019-04-16 | 臻悦生物科技江苏有限公司 | Detect method, apparatus, storage medium, processor and the standardized method of transcript profile data representation amount of Gene Fusion mutation |
| CN110079594A (en) * | 2019-04-22 | 2019-08-02 | 元码基因科技(苏州)有限公司 | High throughput method based on DNA and rna gene abrupt climatic change |
| CN110628880A (en) * | 2019-09-30 | 2019-12-31 | 深圳恒特基因有限公司 | Method for detecting gene variation by synchronously using messenger RNA and genome DNA template |
| CN111118126A (en) * | 2019-11-13 | 2020-05-08 | 上海厦维生物技术有限公司 | mRNA detection method based on high-throughput sequencing |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109637587A (en) * | 2019-01-18 | 2019-04-16 | 臻悦生物科技江苏有限公司 | Detect method, apparatus, storage medium, processor and the standardized method of transcript profile data representation amount of Gene Fusion mutation |
| CN110079594A (en) * | 2019-04-22 | 2019-08-02 | 元码基因科技(苏州)有限公司 | High throughput method based on DNA and rna gene abrupt climatic change |
| CN110628880A (en) * | 2019-09-30 | 2019-12-31 | 深圳恒特基因有限公司 | Method for detecting gene variation by synchronously using messenger RNA and genome DNA template |
| CN111118126A (en) * | 2019-11-13 | 2020-05-08 | 上海厦维生物技术有限公司 | mRNA detection method based on high-throughput sequencing |
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